A Ca(2+)-induced Ca(2+) Release Mechanism Involved in Asynchronous Exocytosis at Frog Motor Nerve Terminals

The extent to which Ca(2+)-induced Ca(2+) release (CICR) affects transmitter release is unknown. Continuous nerve stimulation (20–50 Hz) caused slow transient increases in miniature end-plate potential (MEPP) frequency (MEPP-hump) and intracellular free Ca(2+) ([Ca(2+)](i)) in presynaptic terminals (Ca(2+)-hump) in frog skeletal muscles over a period of minutes in a low Ca(2+), high Mg(2+) solution. Mn(2+) quenched Indo-1 and Fura-2 fluorescence, thus indicating that stimulation was accompanied by opening of voltage-dependent Ca(2+) channels. MEPP-hump depended on extracellular Ca(2+) (0.05–0.2 mM) and stimulation frequency. Both the Ca(2+)- and MEPP-humps were blocked by 8-(N,N-diethylamino)octyl3,4,5-trimethoxybenzoate hydrochloride (TMB-8), ryanodine, and thapsigargin, but enhanced by CN(−). Thus, Ca(2+)-hump is generated by the activation of CICR via ryanodine receptors by Ca(2+) entry, producing MEPP-hump. A short interruption of tetanus (<1 min) during MEPP-hump quickly reduced MEPP frequency to a level attained under the effect of TMB-8 or thapsigargin, while resuming tetanus swiftly raised MEPP frequency to the previous or higher level. Thus, the steady/equilibrium condition balancing CICR and Ca(2+) clearance occurs in nerve terminals with slow changes toward a greater activation of CICR (priming) during the rising phase of MEPP-hump and toward a smaller activation during the decay phase. A short pause applied after the end of MEPP- or Ca(2+)-hump affected little MEPP frequency or [Ca(2+)](i), but caused a quick increase (faster than MEPP- or Ca(2+)-hump) after the pause, whose magnitude increased with an increase in pause duration (<1 min), suggesting that Ca(2+) entry-dependent inactivation, but not depriming process, explains the decay of the humps. The depriming process was seen by giving a much longer pause (>1 min). Thus, ryanodine receptors in frog motor nerve terminals are endowed with Ca(2+) entry-dependent slow priming and fast inactivation mechanisms, as well as Ca(2+) entry-dependent activation, and involved in asynchronous exocytosis. Physiological significance of CICR in presynaptic terminals was discussed.